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Introductory Experiments 71
can be prepared by a simple procedure (see Appendix) sample so that it will layer uniformly into the slots in
that uses detergent lysis and phenol/chloroform extraction the gel. The two dyes serve as visual markers of the
to prepare high quality DNA. Commercially available calf progress of the electrophoresis only and do not stain
thymus DNA can be substituted. Plasmid recombinants DNA or proteins. The purple BPB dye will migrate with
containing E. coli DNA in pUC19 were chosen specifically about twice the relative mobility of the turquoise XC
to work entirely with bacterial DNA and minimize any dye.
misperception of biohazard potential. Competent cells are • TBE electrophoresis buffer: 90 mM TRIS, 2.5 mM
commercially available from several sources, but Na2EDTA, 89 mM boric acid, final pH 8.2 (can be stored
acceptable competent cells can also be prepared with a as a 10x stock).
minimum of expertise. • 1% Agarose: Molten 1% agarose in TBE buffer, heated
to 100oC to melt the agarose and stored in 55oC water
Exercise 1. Gel electrophoresis of nucleic acids bath until needed. Melt the agarose very carefully to
minimize superheating and violoent boiling.
Nucleic acid samples are often subjected to gel • Ethidium bromide (EtBr): For staining gels to visualize
electrophoresis to characterize the size and number of DNA, approximately 200 ml of 4 µg/ml ethidium
different fragments in the sample. In this exercise, DNA bromide in water in a shallow tray, stored covered and
samples that have been digested with restriction enzymes protected from excessive light exposure. EtBr should
and mixed with a tracking dye will be subjected to agarose be treated as a mutagen - a compound capable of
gel electrophoresis. The electrophoresis buffer, the salt causing genetic mutations - and bare skin should not
solution that both conducts electric current and controls come in contact with the solution. It is both
the pH of the solution during the separation of the DNA photosensitive and biodegradable and dilute solutions
fragments, is TRIS/borate/EDTA or TBE, a commonly used are often disposed of via the sink. Concentrated
buffer system. solutions must be saved and disposed of as
When charged molecules are placed in an electric field, biohazardous chemical waste.
the molecules will migrate towards one of the electrodes,
depending on the net charge of the molecule. Nucleic acids Protocol
have an overall negative charge due to the negative 1. Use the molten agarose to pour a minigel as
charges associated with the phosphate backbone of the demonstrated or according to instructions for the gel
molecules, so they will migrate towards the positive unit in use. Illustrations for assembling a generic gel
electrode. Since the distribution of phosphate is very regular unit are included in the Appendix. It is important to be
across the length of the nucleic acid molecule, nucleic acids certain that all small agarose particles are completely
have a constant charge/mass ratio and will therefore melted before use. Allow the gel to completely harden
migrate at the same rate in an electric field. before removing the well-forming comb. This should
Separation of nucleic acid molecules of different size take about 15 minutes.
or conformation is achieved by adding a support matrix to 2. Remove the comb from the solidified gel and transfer
the electric field and forcing the molecules to migrate the gel to a running unit. Submerge the gel in TBE
through this matrix, typically agarose or polyacrylamide gel. buffer.
This matrix acts like a sieve that allows small molecules to 3. Use a manual pipettor with a disposable plastic tip to
go faster than large molecules. The result is that molecules load 10 µl of each sample into one of the wells in the
separate in the matrix according to their relative size and gel. Plasmid and bacteriophage DNA samples should
shape. be easy to load, but chromosomal DNA samples may
This exercise will use gel electrophoresis to examine be quite viscous and difficult to load.
the fragments present in several DNA samples. The 4. Once the samples have been loaded, close the unit
principle goal is to obtain experience in pouring gels, and apply power to the electrophoresis chamber.
loading DNA samples, and visualizing the DNA bands. Typical minigels run at 100-130 volts (60-160
Due to the small volumes of sample to be applied to milliamps), depending on the unit used. During the run,
the gel, samples are routinely handled with a manual the XC and BPB dyes will resolve into two bands, with
micropipet device that uses a disposable plastic tip to the BPB the faster band. Run the gel until this band is
handle a 10 to 20 µl sample. Several commercial devices near the end of the gel, then turn the current off and
are available. A substitute sample loader can be assembled remove the gel.
from a disposable 0.5 or 1 ml syringe fitted with a disposable 5. Transfer the gel to the staining tray containing the
plastic tip (see Appendix). ethidium bromide solution, and stain the gel for 5
minutes. Ethidium bromide is a mutagen, and gloves
Materials should be worn or a spatula used to transfer the gel in
• EcoRI- and HindIII-digested bacteriophage lambda and out of the ethidium solution.
DNA samples containing SM Dye (other DNA samples 6. Transfer the stained gel to a destaining tray containing
can be added or substituted). water. Destain the gel for 5 minutes to remove excess
• Reaction Stop Mix Dye (SM Dye): 10% glycerol, 0.5% ethidium bromide.
SDS (sodium dodecyl sulphate), 0.025% xylene cyanol 7. The stained gel can be visualized with a UV
FF dye (XC), 0.025% bromphenol-blue WS dye (BPB). transilluminator or a UV mineral lamp. DO NOT LOOK
This mix is added to a DNA sample after digestion to DIRECTLY AT THE UV LIGHT!! UV light causes skin
prepare the sample for electrophoresis. The SDS helps burns, is a mutagen, and will cause severe headaches
inactivate DNA binding proteins and releases them from eye damage with direct exposure. Always use a
from the DNA fragments and the glycerol weights the face mask or shield.
Introductory Experiments 73
Materials
• DNA samples prepared in Exercise 2.
• Electrophoresis materials, see Exercise 1.
Protocol
1. Prepare a 1% agarose gel in TBE buffer as in Exercise
1.
2. When the gel has solidified, assemble the gel unit.
Load 15 µl of each of the six samples. You may also
want to include one lane of the digested lambda DNA
used in the first exercise as a DNA standard.
Electrophorese as in Exercise 1.
3. Stain the gel with ethidium bromide and visualize the
DNA bands with a UV light box. A photograph of the
gel is useful, but a sketch of the gel is sufficient for
recording information.
Protocol
1. For each different chromosomal DNA sample, set up
a digestion reaction in a labeled 1.5 ml conical tube
by making the following additions:
Introductory Experiments 75
Protocol
1. Competent cells must be made from a rapidly growing
culture of bacteria to obtain good efficiencies of
transformation. Mid-logarithmic phase of growth (3-5
x 108 cells/ml) is good for preparation of general-use
competent cells. With a fresh overnight innoculation
culture, a 1:50 dilution into a fresh culture will generally
give mid-log cells in 60-90 minutes of growth at 37oC.
If an old overnight culture is used or if insufficient
dilution is made, the bacteria will undergo a significant
lag phase before growth begins.
2. Use sterile techniques to transfer 0.7 ml of the
overnight culture of JM83 to the flask containing 35 ml
of LB. Incubate in a 37oC shaker with vigorous shaking
to allow growth. Monitor the growth of the culture every
20 minutes (A550). When absorbance is approximately
Compare each undigested (-EcoRI) with the corresponding 0.5, the culture is ready to harvest. This should
digested (+EcoRI) sample. Note that the migration position correspond to a mid-logarithmic phase of growth. If a
of the undigested circular plasmid pUC19 DNA (a) changes semi-log plot of A550 versus time is prepared, harvest
when cleaved at the unique EcoRI site to generate a linear of cells should occur in the linear portion of the graph.
molecule (b). The linear undigested lambda DNA (c)
contains five EcoRI cleavage sites, and migrates as six
bands after digestion (two of the bands are of similar size
and can be difficult to resolve, but this double band will be
twice as intense as the other bands) (d). The slowly
migrating, broad, undigested bacterial chromosomal DNA
sample (e) is converted upon digestion to hundreds of
smaller, yet distinct bands (f). Note that as the size of the
genome (pUC19<lambda<E. coli) increases, the number
of cleavage sites also increases, demonstrating that larger
genomes are actually composed of larger amounts of DNA.
8. Add 3.5 ml of cold 30 mM CaCl2, 15% glycerol. This plasmid also contains a genetically engineered portion
GENTLY RESUSPEND THE CELLS. If resuspension of the lac operon of E. coli. When exposed to the inducing
is too harsh, competency may decrease. Return cell compound IPTG, cells containing pUC19 synthesize the
suspension to ice. enzyme ß-galactosidase. This activity can convert the
9. Label 17 sterile microcentrifuge tubes “cJM83”. Aliquot colorless compound X-gal to a blue compound during the
200 µl of competent cells into each tube. Cap tubes. growth of the cells. When the strain JM83(pUC19) is plated
Cells can be used immediately or stored in a freezer on medium containing ampicillin, IPTG and X-gal, the
for future use. To freeze, move the tubes to a pre- resulting colonies will be dark blue in color.
chilled rack in the freezer for storage. Cells can be The plasmid pUC19 was designed with a series of
stored at -20oC (normal lab freezer) for several weeks unique restriction cleavage sites within the coding region
or at -70 oC for months without significant loss of of the ß-galactosidase gene. This region, designated the
competency. Recombination deficient strains (recA- multiple cloning site or MCS, is used for the specific
strains, in particular) rapidly lose viability/competency insertion or cloning of extra DNA fragments. When a
when stored in a freezer. fragment is inserted, the β-galactosidase gene is
10. If competent cells are made and stored for use in a interrupted and the enzyme can no longer be synthesized.
lab exercise, it is wise to transform one tube with a Thus, a strain containing pUC19 with an extra DNA insert,
pure plasmid DNA sample prior to use to verify that designated JM83(pUC19:insert), will still form colonies in
the cells have not lost competency during storage. the presence of ampicillin, but in the presence of IPTG
Larger numbers of competent cells can be prepared and X-gal the colonies will remain white rather than blue.
by scaling all procedures up as needed. When culture Thus, bacteria containing the wild-type pUC19 vector can
volumes are increased, be certain that the culture has be distinguished from cells containing a recombinant
a large surface area to maintain adequate aeration in pUC19 plasmid with an extra DNA insert.
the growing culture. To transform cells with pUC19, the competent cells
will be placed at 43oC, then directly plated on nutrient plates
containing 100 ug ampicillin/ml as a selective antibiotic.
Exercise 4. Transformation of competent cells with Incubation of the plates at 37oC will allow colonies to form.
pUC19 and pUC19 recombinants The presence of the compounds IPTG and X-gal will be
used to distinguish the original vector from recombinant
DNA molecules can be inserted into bacteria by first derivatives containing DNA inserts. The wild-type vector
chemically treating the bacteria to make them competent, will cause the formation of blue colonies and recombinant
or able to take up DNA. This chemical treatment is often derivatives present in the pUC19:E. coli DNA library will
accomplished by exposing the bacteria to specific salts cause the formation of white colonies.
that cause the bacteria to swell and become capable of
binding DNA. Treatment of E. coli with CaCl 2 gives Flow chart, Exercise 4
reproducible transformation results, although higher levels
of competency can be attained with other procedures. After
exposing the competent cells to DNA on ice, a 43oC heat
shock is required to make the competent cells take up the
bound DNA.
In this exercise, E. coli strain JM83 will be transformed
with the plasmid pUC19. This plasmid contains genes that
allow its own replication in the bacterium and contains a
gene (bla) that encodes ß-lactamase, an enzyme that
confers resistance to ampicillin. The host strain JM83 is
sensitive to and will not grow in the presence of ampicillin,
while JM83 containing pUC19, indicated by JM83(pUC19),
will grow and form colonies in the presence of the antibiotic.
Materials
• Plasmid pUC19 vector DNA: 100 µg/ml.
• pUC19:E. coli library DNA: 100 µg/ml.
• Competent E. coli cells: CaCl2 treated competent JM83
cells, 100 µl aliquots, stored -20oC.
• Sterile dH2O.
• LB agar plates containing ampicillin for selection of
pUC19 transformants: 1% Bacto Tryptone, 0.5% Bacto
Yeast Extract, 0.5% NaCl, 1.5% Bacto agar, pH 7.0-
7.2. Autoclave to sterilize, cool to 50oC, add ampicillin
to 100 µg/ml, pour petri plates, and cool. Other
complete E. coli nutrient media can be substituted, as
long as ampicillin is present. Do not use minimal media,
as most laboratory strains of bacteria have several
nutritional deficiencies that require specific
supplements that are generally present in rich media.
Introductory Experiments 77
• 10 mM IPTG: 23.8 mg IPTG (isopropyl-β-D- the number of colonies on a plate and dividing by the
thiogalactopyranoside)/ml dH2O. amount of transforming DNA represented on the plate,
• 5% X-gal: 50 mg X-Gal (5-bromo-4-chloro-3-indolyl- it possible to calculate the efficiency of transformation
β-D- galactopyranoside)/ml dimethylformamide. in terms of transformants/µg DNA. Example: 1 µg of
• 43oC temperature block or water bath. pUC19 DNA was added to 100 µl of competent cells,
• 37oC incubator. and 1 µl of this mix was plated on an LB-Ap plate,
resulting in 227 colonies. The amount of DNA
Protocol represented on the plate is 10 µl/110 µl x 1 µg = 0.1
1. Prepare six LB-Ap plates for transformation by adding µg. Transformation efficiency is 227 colonies/0.1 µg,
IPTG and X-gal to each plate. To a sterile tube add 60 or 2.27 x 103 transformants/µg DNA.
µl 10 mM IPTG and 300 µl 5% X-gal. Mix solution. • JM83 transformed with pUC19:E. coli library should
Place 60 µl of this IPTG/X-gal mix on the surface of give similar numbers of colonies when compared with
an LB-Ap plate. Dip a glass spreader in 95% ethanol the pUC19 transformation, but only a percentage of
and flame to sterilize. Use the spreader to distribute the colonies will be blue. The blue colonies are the
the IPTG/X-gal mix evenly over the surface of the plate. result of transformation with pUC19 containing no
Replace the lid on the plate and invert the plate. Repeat additional DNA insert, and the white colonies are
this process for each of the remaining 5 plates. If you caused by transformation with recombinant pUC19
attempt do do all of the plates at the same time, the plasmids. The transformation efficiency can be
solution may soak into the center of the plates and will calculated for the DNA as described above and the
not be uniformly distributed over the surface. Allow percentage of recombinant molecules can be
the solution to soak into the surface of the plates for determined by the calculation:
15-30 minutes (if possible, place the plates in a 37oC
incubator during this time period). 100 x # white colonies/# total colonies.
2. Thaw three tubes of competent cells at room
temperature. As soon as cells are thawed, label one This exercise demonstrates two fundamental principles of
tube “-DNA”, one tube “+pUC19”, and one tube recombinant DNA methods:
“+library” and place all tubes on ice. 1. DNA can be inserted into bacteria to change the
3. To the “-DNA” tube add 10 µl sterile ddH 2O, to the properties of the cells (ampicillin-sensitive cells
“+pUC19” tube add 10 µl (1 µg) of pUC19 DNA, and converted to ampicillin-resistant colonies).
to the “+library” tube add 10 µl of pUC19:EC library 2. DNA fragments can be re-arranged to change the
DNA. Place tubes on ice for at least 15 minutes (length genetic properties of the DNA molecules (blue colonies
of time is not crucial here). This allows DNA to bind to caused by pUC19 are converted to white colonies
the competent cells. While tubes are on ice, adjust a when DNA fragments are inserted into pUC19 DNA).
water bath to 42-43oC. These principles can be further examined by isolation and
4. Heat pulse the cells to cause DNA uptake. Transfer all examination of plasmid DNA present in the transformant
three of the tubes of competent cells containing DNA colonies.
to the 42oC bath for 90 seconds, then transfer to a
room temperature rack. Exercise 5. Transformation of competent cells with
5. Add 10 µl of one of the transformed cell samples to an pUC19 and pBR322 plasmid DNA
LB-Ap-IPTG-Xgal plate and the rest (100 µl) to another
plate. Dip the glass spreader in ethanol, flame, and One of the most important concepts of DNA biology is that
use to distribute the cells evenly over the surface of genes are composed of DNA and that the introduction of
each plate. Invert the plates and label with amount of DNA into a cell can change the physical properties of the
cells and type of DNA (such as 10 µl, pUC19). Repeat cell. This can be illustrated with petri plates containing a
for each of the tubes of transformed cells (you should simple nutrient medium (Luria broth, Tryptone, Nutrient
end up with no tubes of transformed cells left over agar, essentially any medium that will allow the growth of
and have six labeled plates with cells spread on them). E. coli), a small amount of plasmid DNA, and competent
Place plates in a 37oC incubator for 12-18 hours to E. coli bacterial cells.
allow colonies to form and color to develop. Plates This exercise uses E. coli JM83 cells that have been
can be stored in a refrigerator and color will continue made competent, or able to take up DNA. The cells were
to develop. grown to mid-logarithmic phase, then harvested and
resuspended in 30 mM CaCl2. After incubation on ice for
Analysis and significance of results 30 minutes, the cells were again harvested and
After the plates have been allowed to incubate and colonies resuspended in 30 mM CaCl2 with 15% glycerol. Cells were
have formed and colored, compare all the plates. The dispensed into 0.2 ml volumes in sterile 0.5 ml tubes. Each
following results are typical: of these tubes contains sufficient cells for at least 4 different
• JM83 transformed with no DNA should give no distinct transformation reactions. Competent cells may slowly lose
colonies but may have some smearing or poor growth their ability to take up DNA if not stored at -70oC. For this
in areas of dense numbers of cells. reason, the competent cells are shipped in dry ice to arrive
• JM83 transformed with pUC19 DNA should give many the week before the exercise and are stored in a freezer at
distinct colonies, nearly all of which (>99%) should be about -5oC until used.
blue. The occasional white colony will be a contaminant A DNA sample containing two types of pure plasmid
or a mutant derivative of pUC19 in which the ß- DNA, pUC19 and pBR322, at a concentration of 100 µg/
galactosidase gene is no longer active. By counting ml will be used to demonstrate that DNA can change cellular
78 Tait
properties. The plasmid pUC19, a common plasmid cloning containing pUC19 to form a blue colony - allows rapid
vector used to carry and propagate other DNA fragments sorting of colonies formed by cells containing each of the
in bacteria, has two genes of interest: one confers two types of plasmid. This demonstrates that two plasmids
resistance to the antibiotic ampicillin, and the other encodes that confer the same phenotypic property (such as ability
a ß-galactosidase gene derivative that produces a protein to grow in the presence of ampicillin) can be readily
that can help the bacteria metabolize derivatives of the distiguished if the plasmids differ in ability to confer a
sugar galactose. When the colorless compound X-gal is second phenotype (such as ability to metabolize X-gal to
added to the growth medium, the ß-galactosidase protein a blue compound).
can metabolize the X-gal into a colored derivative that turns This exercise will also demonstrate a second important
the bacterial colonies blue. principle of DNA manipulation: when two genes are both
The second plasmid DNA provided is pBR322, one of present on the same DNA fragment, selection for one of
the older plasmid cloning vectors. Like pUC19, this plasmid the genes will force maintenance of the second gene as
confers resistance to ampicillin, but it also confers well. As the transformed bacteria grow and form colonies
resistance to the protein synthesis inhibitor tetracycline. in the presence of ampicillin but in the absence of
Just like bacteria containing pUC19, bacteria containing tetracycline, there is no selective pressure that requires
pBR322 will be able to grow in medium containing the expression of the tetracycline resistance gene present
ampicillin. Since pBR322 does not contain the ß- on pBR322. Nevertheless, because the tetracycline
galactosidase gene derivative, cells containing this plasmid resistance gene is part of pBR322, growth in the presence
will be unable to metabolize X-gal to a colored compound of ampicillin forces the maintenance of pBR322 and also
and will remain white when exposed to X-gal. Bacteria selects for the covalently attached tetracycline resistance
containing pBR322 will, however, be able to grow in the gene. If the blue and white colonies obtained on the
presence of a concentration of tetracycline that kills cells ampicillin plate containing X-gal and IPTG are transferred
containing pUC19. to a nutrient plate containing tetracycline at a concentration
The bacterial strain JM83 is sensitive to ampicillin and of 20 µg/ml, the blue colonies containing pUC19 will be
will not grow on nutrient plates in the presence of the unable to grow and the white colonies containing pBR322
antibiotic at a concentration of 100 µg/ml. When either will be able to form colonies. This is the fundamental
pUC19 or pBR322 is inserted into the competent JM83 principle that allows a plasmid or a viral DNA molecule to
cells, the cells become transformed and are able to grow be used as a vector to carry and propagate exogenous, or
in the presence of the antibiotic. These two plasmids can extra, DNA fragments. DNA fragments that are covalently
thus cause the same change in bacterial phenotype - ability inserted into a vector by the use of restriction enzymes
to grow in the presence of ampicillin - and this property and DNA ligase will be maintained as the carrier vector
cannot be used to distinguish between cells containing one molecule replicates in a host cell.
of these two plasmids.
Materials
• Three nutrient agar plates containing 100 µg/ml
ampicillin (NA+Ap plates).
• One nutrient agar plates containing 20 µg/ml
tetracycline (NA+Tc plates).
• A water bath or temperature block at 43oC.
• Incubator for plate culture (this can be performed at
room temperature, but growth of colonies will take two
days).
• Glass spreader bar.
• Ethanol or isopropanol for sterilizing glass spreader
bar.
• One 0.2 ml tube of competent JM83 (store these in a
normal freezer [not frost-free if possible] until needed,
then on ice).
• Ten µl of pUC19/pBR322 mixed plasmid DNA at 100
µg/ml.
• 2% X-gal in dimethylformamide (this solvent is
necessary for X-gal).
• 100 mM IPTG in sterile water.
• Sterile toothpicks.
with one another. When incompatible plasmids are inserted DNA into their own genome become resistant to infection
into the same bacterium and the cell is grown in the and may form small colonies within the zone of lysis. The
absence of selective pressure designed to force ability of lambda DNA to cause the lysis of bacteria serves
maintenance of both plasmid types, the plasmids will as the selective marker for the presence of cloning vectors
segregate during division of the transformed bacterium and derived from this bacteriophage. In marked contrast to
the resulting progeny will contain one or the other of the plasmid cloning vectors, which require phenotypic selection
two plasmids. to detect cells containing the vector, the replication of a
lambda cloning vector provides a plaque, the immediate
Exercise 6. Comparison of plasmid and bacteriophage means of detection of cells containing the vector. Because
cloning vectors the lambda plaque is basically a zone of lysed cells,
however, the plaque indicates only where the infected cells
Much of the scientific utility of recombinant DNA technology were. Lambda vectors and recombinants derived from them
is based on the use of self-replicating DNA elements to must generally be maintained not as viable bacterial strains,
serve as carriers for DNA fragments that are not capable but as stocks of virus particles.
of replication in bacteria. Bacterial cloning vectors are An M13 bacteriophage particle contains a single-
based on either bacterial plasmids or bacteriophage. stranded, circular DNA genome of about 8 kilobase pairs.
Plasmids are naturally occurring, circular DNA molecules These particles will only infect host bacteria that are
that can replicate as mini-chromosomes in bacteria. When producing the F-pilus protein, which is part of the mating
isolated from nature, these DNA molecules also generally apparatus encoded by the large F plasmid. Bacteria that
encode resistance to one or more antibiotics and may are male, or F+, can be infected by the M13 phage particle.
contain genes that allow transfer of the plasmid DNA from The entering single-stranded viral DNA is converted to and
one cell to another during bacterial mating. Most cloning replicates as a double-stranded circular DNA. Single-
vectors derived from plasmids retain the plasmid origin of stranded progeny phage identical to the original infecting
DNA replication and one or more antibiotic resistance DNA are produced, coated with viral coat proteins, and
genes, such as the ß-lactamase gene, which allows extruded through the bacteria cell wall into the medium
sensitive bacteria to form colonies on solid medium without lysing the host cell. When propagated in a sensitive
containing ampicillin. With plasmid cloning vectors, host grown on the surface of a solid medium, cloning
selection of bacteria containing the vector requires selection vectors derived from M13 will form small, circular zones of
for a phenotype, or physical characteristic, associated with infected cells that are extruding phage particles. In contrast
a gene present on the vector DNA molecule. to plaques formed by lambda vectors, these plaques are
Cloning vectors have also been constructed from cloudy rather than clear and contain viable, infected cells
bacteriophage, viruses that replicate in bacteria. Two types that can be propagated as bacterial strains. While these
of bacteriophage cloning vectors, derived from either the plaques are biologically quite different than those induced
double-stranded, linear DNA bacteriophage lambda or the by strains of lambda, the plaques appear similar to those
malespecific, single-stranded, circular DNA bacteriophage induced by lambda. M13 vectors and recombinants derived
M13, are in common use in E. coli. Detection of cells from them can be maintained as either viable infected
containing either of these types of vector does not require bacteria containing the double-stranded circular replicating
selection for a phenotypic marker, but is based on the ability form of virus DNA or as stocks of viral particles containing
of the bacteriophage to form a plaque in a lawn of host single-stranded circular DNA.
bacteria, a property associated with the replication of the This exercise will use JM101, an F+ strain of E. coli
viral DNA vectors. that will allow growth of plasmids and M13 to illustrate the
Lambda has a large genome (approx. 50 kilobase pairs similarities and differences in the use of these different
of double-stranded DNA) that can, on insertion into a types of cloning vector.
bacterial host, activate either of two sets of viral genes.
One set of genes allows the bacteriophage to undergo a Materials
lytic cycle during which progeny phage are produced and • Six tubes competent JM101 bacteria.
the infected bacterial cell bursts open, releasing the • 11 µl pUC19 DNA, 100 µg/ml (Ampr plasmid vector).
daughter phage into the medium. When activated, the other • 11 µl mp19 DNA, 100 µg/ml (M13 cloning vector).
set of genes causes the bacteriophage DNA to lysogenize, • Two LB plates containing 100 µg/ml ampicillin (LB-
or insert itself into a specific location in the bacterial Ap).
genomic DNA, where it remains silent while the bacterial • Two LB plates (LB).
cell undergoes normal cellular functions. Certain conditions, • Twenty-five ml LB-soft agar (LB containing 0.8% agar).
such as DNA damage, will induce the lysogen, activating • Sterile 5-10 ml glass or plastic tubes.
the lytic genes. The bacteriophage DNA then excises from
the host chromosome, produces progeny, and lyses the Procedure
host cell. When bacteria are grown on the surface of solid 1. Place the tubes of competent cells in an ice bucket to
medium from a low density to a uniform lawn of cells, the thaw. Label the two LB-Ap plates “pUC19 1 µl” and
presence of lambda can be detected by the appearance “pUC19 10 µl”. Label two LB plates “mp19 1 µl” and
of plaques, circular clear zones in the cloudy lawn of host “mp19 10 µl”. Leave the plates at room temperature
bacteria. As the host bacteria grow to form the indicator to warm up for about 10 minutes.
lawn, successive cycles of infection/production of progeny/ 2. When the competent cells have thawed, label the tubes
cell lysis cause the death and lysis of all of bacteria in a as below:
circular zone surrounding even a single lambda
bacteriophage. Cells that are able to lysogenize the lambda
Introductory Experiments 81
Tube 1 pUC19 1 µl DNA fragments to the linearized vector DNA with the
Tube 2 pUC19 10 µl enzyme T4 DNA ligase, followed by insertion of the DNA
Tube 3 mp19 1 µl into a host cell. Because the presence of restriction
Tube 4 mp19 10 µl enzymes can interfere with the ligation process, the
digested DNA samples are generally extracted with phenol/
3. Add the appropriate amount of each of the DNA chloroform to remove protein. Then, because ligation
samples to the labeled tubes of competent cells and activity is not optimal in the buffer used for restriction of
gently mix in. Allow to incubate on ice for 20 minutes. the DNA’s, samples are precipitated with ethanol, washed
4. Label four of the capped sterile glass or plastic tubes with 70% ethanol to remove salts, dried, and resuspended
as below: in ligation buffer. The enzyme T4 DNA ligase, which uses
ATP as a co-factor, is added to seal the nicks in the DNA
Tube 1 pUC19 1 µl molecules and generate recombinant molecules. The
Tube 2 pUC19 10 µl ligated DNA sample can then be used to transform
Tube 3 mp19 1 µl competent cells to give blue and white colonies.
Tube 4 mp19 10 µl The blue and white colonies obtained after
transformation of cells with a ligated DNA sample can be
5. Use a microwave or boiling water bath to melt the 25 cultured and the plasmid DNA present in the cells extracted
ml of LB-soft agar. Place the melted soft agar in a 55oC and examined. When purifying circular plasmid DNA from
bath for at least 5 minutes to allow the temperature to a bacterial cell, it is necessary to separate the plasmid
decrease from boiling. Use a sterile pipet to dispense away from the chromosomal DNA. Differences in the
3 ml of the liquid agar into the tube labeled “Tube 1 physical properties of these two types of DNA facilitate this
pUC19 1 µl”. Immediately use a pipet device and sterile separation. While purification of large (milligram) amounts
tip to transfer the competent cell/DNA mixture in the of plasmid can be somewhat time-consuming and involve
tube labeled “pUC19 1 µl” into the 3 ml of liquid agar . the use of large cultures (typically 1 liter) of bacteria, a
Pour the mixture onto the surface of the LB-Ap plate variety of rapid isolation procedures allow the purification
labeled “pUC19 1 µl” and gently rotate the plate to of a small amount of plasmid DNA (1- 5 µg) from a small
distribute evenly over the surface of the plate. Carefully culture (1-10 ml). The DNA obtained by these small-volume,
place the overlaid plate aside to solidify and do not rapid-isolation methods, generally referred to as
move it for 5 minutes. If this process is too slow, the “miniscreens” or “minipreps”, is not as pure as that
agar will solidify in the tube or in lumps on the surface obtained by more elaborate methods, but is suitable for
of the plate. If the overlaid plate is inverted too soon many characterization and cloning procedures.
after the pouring process, the soft agar will slide off of These miniscreen procedures arose from the need to
the agar plate. quickly sort through a number of bacterial transformants
6. Repeat this overlay for each of the remaining tubes of that contained recombinant DNA molecules of potential
competent cells. interest. A typical gene isolation experiment might generate
7. Incubate the plates at 37oC to allow growth of the 20 candidates for the desired gene construction. The time,
bacteria. expense and labor involved in purifying the DNA from a 1
liter culture of each candidate were found to be quite
Analysis of results annoying, and abbreviated protocols were developed to
Following 12-18 hours of incubation at 37oC, the bacteria allow the simultaneous screening of many candidates.
will have grown sufficiently to compare the difference in Rapid miniscreen procedures take advantage of the
the two types of vectors. For the cells transformed with different purification properties of chromosomal DNA and
pUC19, the ampicillin in the LB plate rapidly diffuses into supercoiled plasmid DNA to obtain a partial purification of
the soft agar overlay and retards the growth of the non- the plasmid from a small culture. Steps of the process are
transformed cells. Cells that contain the plasmid are generally performed in 1.5 ml plastic tubes, and
resistant to the antibiotic and form small colonies in and centrifugation is in a microcentrifuge that can spin 12-24
on the surface of the soft agar overlay. tubes at 12,000 to 16,000x gravity. Spins used to remove
For each of the bacteriophage vector transformations, precipitates are on the order of 5-15 minutes, and the entire
the non-transformed bacteria will have formed a fairly miniscreen process will take about 90 minutes to purify
uniform layer of cells called a lawn. The M13 mp19 vector the plasmid DNA from 12 samples. These protocols
will cause numerous holes and depressions in the lawn. facilitate both the mass screening of transformants
These are best viewed by holding the plate up to a bright containing DNA molecules of potential interest and the rapid
light. analysis and manipulation of DNA samples of particular
Note that as the size of the vector increases, the interest.
number of transformants decreases (# pUC># mp19). This Following transformation of JM83 with the pUC19:E.
is principally caused by the difference in the sizes of these coli library DNA, incubation of the LB-Ap plates for 12-18
vectors (about 3 kb and 8 kb). hr should result in the formation of bacterial colonies on
the surface of the plates. Those transformants that contain
Exercise 7. The miniscreen: Rapid isolation of plasmid only pUC19 are both Apr and able to metabolize X-gal to a
DNA blue compound and form blue colonies, while those cells
that contain pUC19 plasmids with DNA inserts are Apr but
The construction of a library of recombinant plasmids unable to metabolize X-gal and therefore form white
involves the digestion of chromosomal and vector DNA colonies. The ratio of white to blue colonies is an indicator
with a restriction enzyme, followed by joining chromosomal of the frequency of recombinant transformants in the
population.
82 Tait
To characterize the plasmids in the recombinant RNase stock. Invert to mix. Incubate 10 min at 37oC.
transformants, white colonies are picked with a sterile loop 8. Add an equal volume (approx. 700 µl, simply fill the
and transferred into 2 ml cultures of LB medium, allowed remainder of the space in the tube) of isopropanol.
to grow for 6-18 hr at 37oC with shaking, then subjected to Invert tubes several times to mix. Immediately
a miniscreen procedure. These cultures can either be centrifuge 5 min at room temp in a microcentrifuge.
inoculated the day before needed or grown several days Pour off and discard supernatants.
in advance and stored in a refrigerator until needed. Long 9. Wash DNA pellets by adding 1 ml of 70% ethanol to
term storage of cells for DNA extraction is best each tube. Invert several times to mix. Centrifuge 3
accomplished by harvesting the cells from the cultures (step min at room temp. Pour off ethanol and use a Kimwipe
1 below), adding the SET buffer, and freezing the cell to dry the lip of each tube. Vacuum dry the DNA pellets.
pellets. To proceed with the miniscreen, thaw the pellets Resuspend each pellet in 20 µl dH2O. After DNA has
and vortex to resuspend the cells, then resume with step been allowed to resuspend for 10 minutes on ice, tap
3. the tubes gently to help resuspend the DNA, then
centrifuge 20 seconds to collect solution in the bottom
Materials of the tubes. The DNA is now ready to be digested
• LB medium: 1% Bacto Tryptone, 0.5% Bacto Yeast with restriction enzymes or can be stored frozen until
Extract, 0.5% NaCl, pH 7.0-7.2 (sterilized) for further use.
propagation of cultures to be miniscreened. Any
complete nutrient medium can be substituted. Flow chart, Exercises 7 and 8
• One 2 ml LB E. coli JM83 culture, grown at 37oC with
shaking for 8-15 hours.
• One 2 ml LB E. coli JM83 culture containing the
plasmid pUC19, grown at 37oC with shaking for 8-15
hours.
• Four 2 ml LB E. coli JM83 cultures from white
recombinant colonies obtained from the pUC19:E. coli
library, grown at 37oC with shaking for 8-15 hours.
• SET buffer: 20% sucrose, 50 mM TRIS-HCl pH 7.6,
50 mM EDTA.
• Lytic mix: 1% SDS, 0.2 N NaOH.
• Sodium (Na) acetate: 3.0 M, pH 4.8. Make 3 M acetic
acid and 3 M Na acetate, then mix to pH 4.8. If not
made this way, miniscreens will not necessarily work
well. Store at 4oC.
• RNase stock: Pancreatic ribonuclease A (RNase A), 1
mg/ml in 0.1 M sodium acetate, 0.3 mM EDTA.
• Isopropanol: Room temp.
• Ethanol: 70%, room temp. (70% isopropanol can be
substituted).
• Water (dH2O): Sterile, deionized or distilled.
• Ice.
• Conical microcentrifuge tubes and pipet tips.
Protocol
1. Transfer 1.5 ml of each culture to a labeled
microcentrifuge tube. You should have a total of six
tubes. Spin 1 min to pellet cells. Pour off and discard
supernatants.
2. Resuspend cell pellets in 150 µl of SET buffer. Vortex
or agitate to resuspend cells.
3. To each tube add 350 µl of Lytic mix. Invert several
times to mix. Cells will lyse and solution will clear
slightly. Viscosity increases.
4. Place in ice bath and chill approx. 10 min. Solution
will begin to cloud as SDS precipitates.
5. To each tube, add 250 µl of cold Na acetate buffer. Exercise 8. Characterization of pUC19 recombinant
Invert to mix. Return tubes to ice bath and incubate DNAs
15 min. SDS and chromosomal DNA will precipitate
during this incubation. The miniscreen DNAs will now be digested with restriction
6. Centrifuge tubes 10 min at 4oC in a microcentrifuge. endonuclease EcoRI, the same enzyme that was used to
Pour supernatants (approx. 700 µl) into clean, labeled construct the library. Any DNA fragments that were inserted
microcentrifuge tubes. Discard tubes containing the in the unique EcoRI site of pUC19 will be released upon
pellets. digestion, and agarose gel electrophoresis will allow
7. To each tube containing a supernatant, add 2 µl of characterization of the size and number of additional
fragments.
Introductory Experiments 83
Label six tubes for the digestion reactions, and Many of the gel artefacts commonly associated with
dispense 18 ul of this mix into each tube. Then add 2 miniscreen DNA are illustrated below:
µl of a miniscreen DNA to each of the tubes, for a total
of six different reactions. This is more convenient than
setting up six individual reactions.
2. Incubate at 37oC for 30-60 min, then remove 8 µl from
each digestion and place on a Parafilm strip. Add 3 µl
of SM dye to each sample.
3. Subject samples to electrophoresis on a 1% agarose
gel in TBE buffer. Run a sample of EcoRI- or HindIII-
digested lambda DNA as a molecular weight standard.
4. Following electrophoresis, staining and visualization
of the DNA fragments will reveal any additional
fragments present in the pUC19 recombinants. While
a photographic record of the gel allows convenient
measurement of fragment position for the purpose of
calculating mobilities, and hence determining
molecular weights, photos are expensive. A ruler can
be laid next to the gel on the UV box, and a drawing Lane a. Undigested DNA with both supercoiled (dark
with measurements added used to construct a graph bands) and open circular (stippled bands) forms.
of fragment mobilities. Lane b. Partially digested plasmid DNA sample with insert
(bottom band), plasmid vector (second from bottom), linear
Analysis and significance of results recombinant cut at only one of two cleavage sites (third
A considerable amount of sample variability is common from bottom), and open circular DNA (stippled band).
when learning to prepare miniscreen DNA. A typical gel Lane c. Digested plasmid DNA sample containing
with good quality DNA is illustrated below: undigested RNA (stippled region).
Lane d. Digested plasmid DNA sample contaminated with
digested bacterial chromosomal DNA not eliminated during
the DNA preparation.
Lane e. Unmelted agarose specks causing DNA streaking
and spots in the gel.
Lane f. Too much DNA loaded in the lane causing distortion
of bands.
Lane g. Bubble in sample well deforming DNA bands.
Lane h. Digested DNA sample degraded by contaminating
exonuclease or endonuclease.
approach called DNA fingerprinting. This method takes fingerprint of each individual:
advantage of the fact that a single nucleotide sequence
difference can cause the appearance or disappearance of Mother = 100 µl H + 100 µl H (HH)
a restriction enzyme cleavage site in DNA. The individual- Baby = 100 µl H + 100 µl E (HE)
specific differences in restriction enzyme cleavage site Rock Star = 100 µl H + 100 µl K (HK)
position relative to a specific gene can cause variation in Ex-drummer = 100 µl S + 100 µl B (SB)
the size of the DNA restriction fragment that carries that Mailman = 100 µl S + 100 µl E (SE)
gene in different individuals. The variation in the size of a
restriction fragment that carries a specific gene, referred Loading 7 µl of each mix will deliver 350 ng of DNA, giving
to as “restriction fragment length polymorphism” (RFLP), distinctive diploid DNA fingerprint patterns for each
can be used to genetically match DNA samples with the individual.
donors from which they were obtained. Individuals who
appear identical for a specific physical trait can be easily • Gel box, gel tray and comb.
distinguished if sequence differences that cause changes • Power supply.
in DNA restriction fragment sizes are associated with that • UV light source.
trait. • Dry agarose.
When applied to identification of human DNA samples, • 10 mg/ml ethidium bromide.
DNA fingerprint analysis is complicated by the presence • 10X TBE gel buffer.
of hundreds or thousands of DNA fragments following
digestion of DNA samples with a specific restriction Background
enzyme. Rather than attempt to analyze all of the DNA While living with a rock star, a young woman becomes
fragments, the fragments are generally separated by gel pregnant, whereupon the rock star promptly severs his
electrophoresis, transferred to a membrane, and allowed relationship with the woman, leaving her and the resultant
to react with a DNA or RNA hybridization probe that will baby completely penniless. The mother of the child sues
specifically anneal to a small number of the many different for child support, claiming the rock star as the father of her
DNA fragments. The probe fragment generally contains a baby. The following are the summary statements from each
nucleoside that is either radioactive (such as 32PdATP) and of the individuals who become involved in the resulting
can be detected by autoradiography or has been modified paternity suit:
(such as biotinylated dATP) to be recognized by specific
antibodies and detected by a color reaction. The image Mother: Rock Star is the father of my baby. He should
that is obtained by visualization of the probe is a subset of be required to financially support us.
the total set of DNA fragments and can be referred to as a Baby: Wah! Burp.
DNA fingerprint. When hybridization probes are chosen Rock Star: The child is not my son. The males in my
carefully, the fingerprints can be specific for DNA that has family were found through genetic analysis
been found to have a high degree of individual-specific to carry a recessive gene that causes
variation. When sufficient information has been obtained incurable craving for disco music. On the
about the frequency of occurence of a specific DNA advice of my doctor, I had a vasectomy when
fingerprint pattern, the DNA fingerprints of two DNA I was twenty-three to prevent transmission
samples can be used to calculate the probability of whether of this genetic disorder to my children. I
the two DNA samples were obtained from the same kicked her out because I caught her in bed
individual. with my Drummer, not because she was
Since humans are diploid and contain two copies of pregnant. She should talk to him about
each chromosome, one from the mother and the other from support.
the father, a human DNA fingerprint can be thought of as Ex-drummer: Who says I’m the father? I wasn’t the only
the combination of two fingerprint patterns. Since these other guy she was seeing. I even caught the
patterns can be used to link an “unknown” DNA sample to mailman in the bedroom with her. I couldn’t
DNA samples obtained from specific individuals, DNA support her anyways. I can’t get work since
fingerprint analysis is becoming increasingly common as Rock Star kicked me out of his band and I
courtroom evidence in paternity suits and in criminal got busted with 2 pounds of heroin.
prosecution involving crimes like rape and murder. This Mailman: Sure I was in her bedroom. I had a package
exercise will use bacteriophage lambda DNA digested with for Rock Star and needed a signature. When
various restriction enzymes to simulate the results of a DNA I rang the doorbell, she called out she was
fingerprint analysis and help determine paternity of a child. sick in bed, but the door was unlocked and
she would sign for the package if I didn’t
Materials mind risking the flu. I was leaving her
Bacteriophage lambda DNA at a final concentration of 50 bedroom when Drummer walked in the front
µg/ml in gel loading buffer, digested with the following door. I think the door was unlocked and she
restriction enzymes: HindIII (H), EcoRI (E), BamHI (B), KpnI was in bed waiting for Drummer.
(K), and SalI (S). Each of these digests will be used to
simulate a haploid DNA fingerprint (one set of At the request of the court, DNA is extracted from blood
chromosomes). To make the test DNA samples obtained samples obtained from each of these individuals and
from the individuals involved in the paternity suit, the provided to you, who will perform the fingerprint analysis
digested lambda DNA samples have been mixed in the and interpret the results.
following manner to simulate the diploid chromosomal
Introductory Experiments 85
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